York at Buffalo, Buffalo, New York 14214 labile than the phosphatase, thus permitting the latter to be studied independently of the former (13). Chen and Charalampous (7) have achieved separation of these activities on DEAE'-cellulose using the yeast system. Partial purification of the cyclizing enzyme and related studies on cell-free systems from rat testis, yeast, and Neurospora have generated a wealth of information regarding the mechanism of action (3,8,13,16,27,31), physical and chemical properties of the soluble system (2, 5, 6, 12), and identity of substrate, intermediates, and product (4,6,13,15).In plants, cyclization of -glucose 6-P to myoinositol takes on added significance in light of the observation that oxidative cleavage of myoinositol to n-glucuronate and conversion of the latter to uronate and pentose components of cell wall polysaccharides provides an alternative route from 1-glucose to products of D-glucuronate metabolism that bypasses the oxidation of UDP-D-glucose (22). Further evidence of the prevalence of the cyclizing enzyme in plants and a comparative study of its properties and mechanism with those occurring in yeast and rat testis is desirable. We have conducted such a survey in the search for a dependable plant source of this enzyme. On the basis of this search we have chosen to isolate and purify the cycloaldolase from suspension cell cultures of Acer pseudoplantanus L.In 1962 Loewus and Kelly (23) demonstrated the cyclization of 1-glucose to myoinositol in detached parsley leaves. Since then, the enzyme system catalyzing this conversion has been isolated from several sources. 1-Glucose 6-P is the actual substrate (4). Cell-free preparations from rat testis (14) and Candida utilis (4) revealed that D-glucose 6-P is converted to myoinositol in two enzymatic steps; first, cyclization of the sugar phosphate to 1-L-myoinositol 1-P (7, 15), then hydrolysis of 1-L-myoinositol 1-P to myoinositol and P1 (6, 13). The crude systems required both NAD+ and Mge for activity, but resolution of the cycloaldolase from the phosphatase activity revealed that only NAD+ is needed by the cyclizing enzyme, and the Mg'+ requirement is associated with the phosphatase. Similar systems have been found in Neurospora, Oryza sativa, Phaseolus vulgaris, and Sinapis alba (19,28,30).In rat testis preparations, the cycloaldolase is more heat
MATERIAL AND METHODSHerbaceous plants were grown under green house conditions. Leaves were harvested just prior to use.A sample of pollen (Lilium longiflorum, cv. Croft) that had been held in storage at -20 C for several months was freed of "pollen kitt," a gummy residue adhering to pollen grains, by rinsing in medium prepared according to Dickinson (11). Washed grains were divided into two portions, one which was homogenized immediately and the other after incubation for 1 hr at 25 C in fresh medium. Pollen tubes were not microscopically visible at the end of 1 hr, but prolonged incubation (6-8 hr) of separate aliquots showed about 70% germination.Corn seeds (Zea mays, Agway h...
